The long-term objective of this proposal is to investigate the mechanism that regulate the productive life cycle of oncogenic human papillomaviruses (HPV). Papillomaviruses are small DNA viruses which induce a variety of proliferative lesions in most mammals including humans. Of the 71 types of human papillomaviruses that have been identified, a subset are associated at a high frequency with anogenital cancers and these are referred to as the high risk or oncogenic types (HPV 16, 18, 31, 33, and 51). Viral production occurs in low grade lesions which are only slightly altered in their pattern of differentiation from that seen in normal cells. The production of viral particles, genome amplification, capsid protein synthesis, and virion assembly is dependent upon differentiation and is restricted to suprabasal cells. In carcinomas, viral DNA is usually found integrated into host chromosome and no viral production is seen. Several years ago, we first used an in vitro system (raft cultures) for the stratification and differentiation of keratinocytes at the air-liquid interface to duplicate the life cycle of human papillomaviruses in tissue culture. When the CIN 612 cell line, which was derived from a patient biopsy and found to stably maintain episomal copies of HPV 31, was grown in raft cultures, the amplification of viral DNA was detected in suprabasal cells. Furthermore, upon the addition of phorbol esters to the growth media, the induction of late gene expression and virion production was observed. This system was then used to characterize the patterns of differentiation-dependent viral gene expression. Recently, we have extended these studies and synthesized HPV 31 in raft cultures from transfected cloned DNA templates. Using mutants in E1 which fail to establish episomal genomes, a requirement of extrachromosomal templates in the induction of the late gene expression was demonstrated. These methods provide the opportunity for a detailed genetic analysis of HPV functions. In this application, we propose to extend our studies on viral pathogenesis and perform a genetic analysis of the requirements for viral replication during the vegetative life cycle. The following specific aims are proposed: 1) Which cis sequences necessary for maintenance and amplification of HPV 31? 2) How do the known viral replication factors, E1 and E2, function in viral pathogenesis? 3) What signals activate the differentiation-dependent amplification of viral genomes?